The overall goal of this research program is to discovery new natural products that will lead to novel anti-cancer drugs. This will be accomplished by using two powerful new molecular genetic approaches for creation of complex natural products. First, combinatorial biology libraries will be made by harnessing extensive metabolic potential directly from culturable marine microorganisms. A sophisticated gene cloning and pathway manipulation system using cosmid and bacterial artificial chromosome-based vector systems will be used to generate natural products through heterologous expression in Streptomyces venzuelae. Microbial genomes from marine actinomycetes (provided by Wright and Crews), cyanobacteria (provided by Gerwick), myxobacteria and fungi (provided by Crews) will be accessed and libraries enriched for secondary metabolite biosynthetic gene clusters will be generated to express diverse natural product biosynthetic pathways for subsequent analysis in a broad array of "smart assays" developed at Novartis Pharmaceuticals (Bair). Second, combinatorial biosynthesis approaches will be used to engineer specific changes to biosynthetic pathways from the curacin A (Gerwick) and latrunculin (Crews) polyketide systems. In this work, polyketide synthase gene probes based on highly conserved sequence motifs will be used to isolate individual sets of overlapping cosmid clones that comprise the complete curacin A and latrunculin biosynthetic gene clusters for subsequent characterization at the molecular genetic level. This information is required to generate a wide range of molecular modifications for these important structures that have already been identified as promising anti-cancer drug leads. Generation of novel structures based on the curacin A and latrunculin core molecules will be accomplished through deliberate modification of genes and enzymes that specify choice of loading domain, extender units, keto group processing and termination of the curacin A and latrunculin chain elongation intermediates. A parallel effort will be pursued to discover, evaluate and engineer novel forms of natural product tailoring enzymes with flexible substrate specificity. Overall, we expect to generate a large number of exciting new natural products for development as novel anti-cancer agents using the powerful tools of microbial genomics, combinatorial biology, combinatorial biosynthesis, natural products chemistry and innovative anti-cancer drug screening assays systems.